Methods of using cellulase for reducing the viscosity of feedstock

ABSTRACT

The invention provides methods for treatment of feedstock to reduce the relative viscosity and promote release of fermentable sugars.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase, pursuant to 35 U.S.C. §371,of International application Ser. No. PCT/US2011/027923, filed Mar. 10,2011, designating the United States and published in English on Sep. 15,2011 as publication WO 2011/112824 A1, which claims priority to U.S.provisional application Ser. No. 61/312,636, filed Mar. 10, 2010. Theentire contents of the aforementioned patent applications areincorporated herein by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported by the U.S. Department of Energy, Grant numberDE-FG36-08GO88142. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Lignocellulosic biomass (LCB) could serve as a primary carbohydratefeedstock to partially replace petroleum-based fuels and chemicals. LCBof terrestrial plants is composed of the thermoplastic lignin (15-25%)and two carbohydrate polymers, cellulose (35-50%) and hemicellulose(20-35%). Processes for depolymerization and fermentation of LCB aremore complex and more capital intensive than established technologiesfor cornstarch or cane syrup. Unlike the starch, LCB has been designedby nature to serve as a structural element that resists microbialdeconstruction. Pretreatments such as dilute mineral acids or basetreatments are needed to render cellulose polymers accessible toenzymatic attack. Steam treatment with dilute mineral acids hydrolyzeshemicellulose into a pentose-rich syrup. This process is accompanied byside reactions and the production of inhibitors (furans, organic acids,and phenolics) that retard fermentation.

SUMMARY OF THE INVENTION

The invention provides methods for decreasing the relative viscosity offeedstock using chemical and enzymatic methods.

In one aspect, the invention generally provides a method of decreasingthe relative viscosity of feedstock, the method involving incubating thefeedstock with about 0.01 FPU to about 20 FPU cellulase/g dry weight offeedstock for about 10 minutes to about 10 hours at a pH of about 2 toabout 6 and at a temperature of about 50° C. to about 70° C.; therebydecreasing the relative viscosity of the feedstock.

In another aspect, the invention provides a method of decreasing therelative viscosity of feedstock, the method involving incubating thefeedstock with about 0.50 FPU to about 5 FPU cellulase/g dry weight offeedstock cellulase for about 15 minutes to about 2 hours; where thefeedstock is incubated at a pH of 3 or less and at a temperature of atleast 60° C.; thereby decreasing the relative viscosity of thefeedstock, where the relative viscosity after treatment is less than1500 cP.

In another aspect, the invention provides a method of reducing theviscosity of a feedstock having a viscosity of at least 20,000 cPinvolving combining the feedstock having a viscosity of at least 20,000cP with a feedstock obtained by treating a feedstock by the method ofany previous aspect, wherein the feedstock having a viscosity of atleast 20,000 cP comprises 30% or less (e.g., 5%, 10%, 15%, 20%, 25%,30%) of the volume of the feedstock treated by the method of anyprevious aspect.

In other embodiments of any of the above aspects, the feedstock is abagasse, corn fiber, corn stover, a plant waste material, or aprocessing or agricultural byproduct. In other embodiments, thefeedstock is a sugar cane, monoenergy cane, sorghum sudan, Miscanthus,switchgrass, wheat, milo, bulgher, barley, rice, corn, beet, or tree. Instill other embodiments, prior to treatment, the feedstock has arelative viscosity of at least 20,000 cP. In various embodiments, therelative viscosity of the feedstock having a relative viscosity of atleast 20,000 cP is reduced by at least 50%, 60%, 70%, 80%, 90%, 92%,95%, 97%, 98%, or 99% as compared to the starting material. In otherembodiments, the relative viscosity after treatment is less than 8000cP, is less than 6000 cP, is less than 3000 cP, or is less than 1500 cP.In still other embodiments, the feedstock is incubated with about 0.05FPU to about 20 FPU cellulase/g dry weight of feedstock (e.g., 0.05,0.1, 0.25, 0.5, 1, 5, 10, 15, and 20), with about 0.50 FPU to about 5FPU cellulase/g dry weight of feedstock, or with about 5 FPU to about 10FPU cellulase/g dry weight of feedstock. In other embodiments, thefeedstock is incubated for about 10 minutes to about 6 hours (e.g., 10,15, 20, 25, 30, 35, 40, 45, 50, and 60 minutes and 1, 1.5, 2, 2.5, 3, 4,5, and 6 hours). In other embodiments of any of the above aspects thefeedstock is incubated for about 15 minutes to about 2 hours. In otherembodiments, the pH is about 2 to about 6 (e.g., 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, and 6), the pH is about 3 to about 6, the pH is about 2 to about5, the pH is about 2 to about 4, or the pH is about 2 to about 3 (e.g.,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3). In still otherembodiments, the feedstock is incubated at a temperature of 40° C. to80° C. (e.g., 40, 50, 60, 70, and 80° C.), is incubated at a temperatureof 45° C. to 80° C., is incubated at a temperature of 50° C. to 80° C.,is incubated at a temperature of 50° C. to 75° C., or is incubated at atemperature at a temperature of 50° C. to 60° C. In still otherembodiments, the feedstock is pretreated in an acidic solution prior toincubation with cellulase. In some embodiments, the acidic solution isphosphoric acid. In other embodiments, the acidic solution is sulfuricacid. The acidic solution contains about 0.01% to about 10% phosphoricacid (e.g., 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%), contains about 1% to about 8%phosphoric acid, or contains about 4% to about 6% phosphoric acid. Inother embodiments, the acidic solution contains about 0.01% to about 10%sulfuric acid (e.g., 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%), contains about 1% to about 8%sulfuric acid, or contains about 4% to about 6% sulfuric acid. Invarious embodiments, the feedstock is pretreated in an acidic solutionfor about 2 minutes to about 2 hours (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 45, 60, and 120 minutes). In some embodiments, the feedstockis pretreated with heat, e.g., with steam, for about 1 hour to about 3hours prior to treatment with cellulose (e.g., 1, 1.5, 2, 2.5, and 3hours). In certain embodiments a treatment with steam is after atreatment with an acidic solution.

In other embodiments of any of the above aspects or of any other aspectof the invention delineated herein, prior to treatment, the feedstockcontains a mixture of about 8-12% dry weight in an aqueous solution(e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, and 12%). In variousembodiments of any of the above aspects, the method extracts at leastabout 40%, 50%, 55%, 60%, or 65% of the fermentable sugars from thefeedstock, where the fermentable sugars are one or more of xylose,galactose, and arabinose. In other embodiments, the amount offermentation inhibitors extracted by the method is less than 1%, lessthan 0.75%, less than 0.5%, or less than 0.2% of the volume of theincubation mixture, where the fermentation inhibitors are one or more offurfural, hydroxymethylfurfural, formate, and acetate.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the effect of phosphoric acid hydrolysate and cellulaseenzyme loading on saccharification and relative viscosity. FIG. 1A showsthe effect of incubation time on relative viscosity (Eqs. 1, 2 and 3).FIG. 1B shows the effect of enzyme loading on relative viscosity andsaccharification (6 hour incubation; Eq. 4 and Eq. 5, respectively).FIG. 1C shows the effect of incubation time on saccharification (Eqs. 6,7 and 8). FIG. 1D shows the effect of incubation time onenzyme-solubilized sugars. Polynomial equations were developed thatdescribed saccharification. The decline in viscosity was modeled as aone phase exponential decay. Confidence limits (dashed lines) have beenincluded for most curves (p<0.05). The thick continuous lines weregenerated using model equations. For saccharification, sugars present atzero time have been subtracted. Reported sugars were produced solely byenzymatic action.

FIG. 2 is a scatter plot of viscosity versus saccharification (10% w/wslurries of acid pretreated fiber in hemicellulose hydrolysate). Sugarspresent at zero time have been subtracted. Reported sugars were producedsolely by enzymatic action. Dashed lines indicate the 95% confidencelimits (p<0.05). The thick continuous line was generated using the modelequation for a one phase exponential decay (Eq. 9). Flow through 12 mmdiameter funnel stems was correlated with a viscosity of 3,000 cP orless.

FIGS. 3A and 3B show the effect of acid pretreated fiber additions onthe viscosity of cellulase-digested slurries containing 10% dry weightacid pretreated fiber. Enzyme-digested slurries of acid pretreated fiberwere prepared by incubating for (a) 2 h or (b) 6 h (Eqs. 10, 11 and 12)and cooled to room temperature to slow enzymatic action. These weremixed with 10% dry weight slurries of acid pretreated fiber that had notbeen treated with enzymes. Viscosities were measured immediately (within1 min) These data were modeled as equations for sigmoid curves (Eqs. 10,11 and 12), shown as thick black lines. Insufficient data points wereavailable to model the 2-h treatment (FIG. 3a ). Values connected withthin solid lines are within range of instrumentation (i.e. <20,000 cP).Curves derived from FIG. 1a were used to estimate the value at theimmeasurable point for each curve and plotted as open symbols connectedby a dashed arrow.

DEFINITIONS

“Bagasse” is the fibrous residue remaining after sugarcane or sorghumstalks are crushed to extract their juice.

As used herein, “centipoises” or “cP” is understood as a measurements ofrelative viscosity were used to compare slurries of pretreated bagassefollowing enzymatic digestion. Due to the nature of the material, a truecP value cannot be determined, however, a cP value for the purpose ofcomparison can be made. The values provided herein were made using aBrookfield DV-II+Pro Viscometer equipped with a T-bar (T-C spindle, 100rpm). Although values are reported as centipoise (cP), these are onlyuseful on a comparative basis between samples discussed herein, orbetween samples discussed herein with samples tested in a comparablemanner. Characterization of the relative viscosity of a mixture hereinis understood to include these limitations.

As used herein, a “percent decrease in relative viscosity” is understoodas equaling 100%−(ending cP)/(starting cP) wherein the viscosity ismeasured by the methods provided herein. When the starting material istoo viscous to be measured using the device provided herein, unlessotherwise stated, the material is arbitrarily assigned a viscosity of20,000 cP for the purpose of making the above calculation.

As used herein, “feedstock” is understood as any plant based materialthat can be converted through a chemical and/or mechanical processesinto a substrate for ethanol production, e.g., fermentable sugars, byfermentation or other methods (e.g., enzymatic methods). Feedstockincludes, but is not limited to a bagasse, corn fiber, corn stover, aplant waste material, a processing or agricultural byproduct, sugarcane, monoenergy cane, sorghum sudan, Miscanthus, switchgrass, wheat,milo, bulgher, barley, rice, corn, beet, or tree.

As used herein, “fermentation inhibitors” are naturally occurringproducts, e.g., acetate, furfural, and formate, that are produced in theprocess of converting a feedstock into a substrate for ethanolfermentation that decrease the efficiency of microbes or enzymes toconvert the substrate for ethanol production into ethanol.

“Filter Paper Unit” or “FPU” as used herein is intended to be defined asset forth by IUPAC. Specifically, FPU is intended to mean the amount ofenzyme in a 0.5 ml aliquot that results in a value of 2.0 mg of reducingsugar as glucose from 50 mg of filter paper (4% conversion) in 60minutes.

As used herein, a “10% slurry” is understood to include about 8% toabout 12% insoluble material per total volume.

Unless otherwise indicated, percent solutions or mixtures are understoodto be weight/volume.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

“At least” a particular value is understood to mean the specific valueprovided, optionally including more.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

DETAILED DESCRIPTION OF THE INVENTION

Options to consolidate bioprocessing steps with lignocellulose arelimited in part by hydrolysate toxicity, material handling associatedwith fibrous suspensions, and the low activity of cellulase enzymes.Combinations of enzyme dose and treatment conditions provided hereinwere shown to improve flow properties and pumping of acid pretreatedsugarcane bagasse slurries (10% dry weight). Low levels of cellulaseenzyme (0.1 and 0.5 FPU/g dry weight acid pretreated bagasse) were foundto reduce the viscosity of these slurries by 77-95% after 6 h ofincubation. This coincided with solubilization of 3.5% of the bagassedry weight. Flow of these slurries through small funnels was found to bean excellent predictor of success with centrifugal and diaphragm pumps.Equations were derived that describe changes in viscosity andsolubilized sugars as a function of time and cellulase enzyme dosage.

When suspensions of fresh acid pretreated bagasse (10% dry weight) wereblended with suspensions of acid pretreated bagasse (10% dry weight)that had been previously digested with cellulase enzymes (lowviscosity), viscosity did not increase linearly. Viscosity of thesemixtures remained relatively constant until a threshold level of freshfiber was reached, followed by a rapid increase with further additions.Up to 35% of fresh acid pretreated bagasse could be blended withenzyme-digested fiber (5.0 FPU/g dry weight acid pretreated fiber; 6 h)with only a modest increase in viscosity. Without being bound bymechanism, a simple model is proposed to explain this phenomenon. Thesmooth surfaces of enzyme-treated fiber are proposed to hinder thefrequency and extent of interactions between fibrils of fresh fiberparticles (acid pretreated) until a threshold concentration is achieved,after which fiber interactions and viscosity increase dramatically.These results were used to model the viscosity in an ideal continuousstirred tank reactor (liquefaction) as a function of residence time andenzyme dosage.

Recent progress has been made in developing pretreatment conditions withphosphoric acid that minimize side products and more robust biocatalyststhat have increased resistance to fermentation inhibitors such asfurans. These improvements could facilitate the simultaneousfermentation of hemicellulose hydrolysate and cellulose-enriched fiberin a single vessel, avoiding a complex and costly liquid-solidseparation. However, physical handling of LCB fiber suspensions remainsa critical issue. Considerable bridging occurs among the fibers ofsugarcane bagasse (dry solid or in an aqueous slurry; with or withoutacid pretreatment) that severely limits mixing and pumping. At 10%solids (dry weight) and higher, fibrous suspensions of acid pretreatedbagasse trap most of the water and pour as a single tangled unit from alaboratory beaker. Warwick et al. (1985) reported that following mildacid treatment of lignocellulose, small molecules penetrate cell wallcapillaries (spaces between microfibrils and cellulose molecules) in theamorphous regions. This external fibrillation greatly increases thewater-holding capacity of LCB slurries by enhancing the abundance ofcapillary-like regions (external surface area) and potential bondingareas between fibrils and fibrils and water. Attempts to pump slurriescontaining 10% solids content or higher at pilot scale typically resultin dewatering and blockage. Although it remains to be seen if thisproblem persists in very large commercial scale plants, mixing andpumping of LCB slurries represent significant challenges during pilottesting and scale-up.

Previous studies have investigated the effects of particle size onrheological properties of red-oak sawdust and the effect of initialsolids loading on power consumption, glucose yield and rheologicalproperties of dilute acid pretreated corn stover slurries. Decreasingthe particle size of red-oak sawdust from 590-850 μm to 33-75 μmappeared to improve the efficiency of enzymatic saccharification by over50% (i.e., conversion of cellulose to glucose) as well as reduceviscosities by as much as 98% using an initial solids concentration of10% (w/w). A study by Rezania et al. (2009) reported that reducing theparticle size of red-oak sawdust to <1 μm by sonication did not improveglucose release by cellulases and increased the viscosity. This increasein viscosity was proposed to result from dominant frictional effects atthe tested particle size range.

Herein, the effects of cellulase treatments on the relative viscosityand flow properties of acid pretreated bagasse fiber (10% dry weightslurries in hemicellulose hydrolysate) without particle size reductionhave been analyzed. Relative viscosity (single phase exponential decay)was correlated with the extent of saccharification under a wide range ofconditions. These results were used to model viscosity changes in anideal continuous stirred tank reactor with different amounts ofcellulase.

As demonstrated herein, it was unexpected that low levels of cellulaseenzymes were sufficient to reduce viscosity and improve the flowproperties of acid pretreated bagasse slurries. Relationships betweenviscosity, cellulase dosage, incubation time, and saccharification weremodeled and correlated with the ability of slurries to flow throughgraded funnels and be effectively pumped. Methods are provided to fordecreasing viscosity at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%,98%, or 99% as compared to the starting material, while extracting atleast about 40%, 50%, 55%, 60%, or 65% of the sugars (e.g., xylose,galactose, and arabinose) from the feedstock and limiting production offermentation inhibitors (less than 1%, less than 0.75%, less than 0.5%,or less than 0.2%). It was also unexpected that the addition of acidpretreated fiber slurries to enzyme digested (and acid pretreated)slurries had little effect on viscosity until a threshold concentrationwas reached, after which viscosity increased rapidly. A simple model wasproposed to explain this phenomenon. Data from this study were used tomodel viscosity changes in an ideal continuous stirred tank reactor(CSTR).

Example 1 Materials and Methods Materials

Sugarcane bagasse was provided by the Florida Crystals Corporation(Okeelanta, Fla.). Kerry Biocellulase W (164 mg protein/ml;approximately 50 filter paper units/ml) was provided by KerryBiosciences (Cork, Ireland). Novozyme 188 β-glucosidase (approximately277 cellobiase units/ml) was purchased from Sigma-Aldrich (St. Louis,Mo.). Thymol, phosphoric acid, and other chemicals were purchased fromThermo-Fisher Scientific (Waltham, Mass.).

Dilute Acid Pretreatment of Sugarcane Bagasse

Sugarcane bagasse was received at approximately 50% moisture. Bagassewas soaked in a 10-fold excess of 1% (w/w) phosphoric acid (2 h) anddewatered to 33% dry weight using a Centra CF basket centrifuge(International Equipment Company, Needham Heights, Mass.; 4 minutes at3000 rpm). This acid-impregnated bagasse was autoclaved (500 g batchesdivided among three 1-L Pyrex beakers) at 145° C. (1 h), and cooled toroom temperature. Sufficient deionized water was added to adjust thetotal weight to 3 kg (6 times the initial dry weight of untreatedbagasse). The resulting slurry contained a total of 167 g/L acidpretreated bagasse fiber (approximately 112 g insoluble fiber/litervolume). After soaking and manual mixing to allow equilibration (2 h),most of the hydrolysate was removed by centrifugation. Resulting acidpretreated fiber (33% dry weight) and hemicellulose hydrolysate werestored at 4° C. Samples of acid pretreated fiber were washed with waterprior to carbohydrate analysis.

Saccharification with Biocellulase W and β-Glucosidase

Saccharification of acid pretreated fiber was tested using KerryBiocellulase W and Novozyme 188 β-glucosidase. Samples of acidpretreated fiber (20 g) were added to 500-ml flasks containing thymol(10 mg) as a preservative. Sufficient pH-adjusted hydrolysate (pH 3-8)containing Kerry Biocellulase W (0-5.0 FPU/g dry weight acid pretreatedfiber) and Novozyme 188 β-glucosidase (0-3 cellobiose units/g dry weightacid pretreated fiber) was added to adjust the contents to 200 g (10%dry weight acid pretreated fiber). Most experiments were conducted at pH5.0 (adjusted with 45% KOH) and 55° C.

Flasks with enzymes and acid pretreated fiber were incubated at 300 rpm(25-80° C.) for 1 h, and at 200 rpm thereafter. After adjustment forevaporative loss with deionized water, samples were removed and storedat −20° C. until analyzed. The extent of saccharification was measuredas enzyme-solubilized sugar by the subtraction of sugars present beforeenzyme addition.

Relative Viscosity Measurement

Measurements of relative viscosity were used to compare rheologicalproperties of acid pretreated fiber slurries following enzymaticdigestion using a Brookfield DV-II+Pro Viscometer equipped with a T-bar(T-C spindle, 100 rpm). Although values are reported as centipoise (cP),these are only useful on a comparative basis. The non-Newtonianproperties of this fibrous material preclude a more rigorousinterpretation. Values ≧20,000 cP represent near immobilization of thespindle. The relative viscosity of water, acetate buffer andhemicellulose hydrolysate were also measured for comparison(approximately 1 cP for all).

Flow Properties Tests Using Graded Funnels

Plastic laboratory funnels with internal stem diameters 7 mm, 12 mm and17 mm were used to compare the flow properties of acid pretreated fiberafter the various treatments with Kerry Biocellulase W. Flow wasassisted by gentle tapping. The slurries (10% dry weight acid pretreatedfiber) were found to either pass through the funnel (indicated by a Y inTable 2) or to form a plug that resisted flow (indicated by an N).

Carbohydrate Composition and Analyses

Carbohydrate compositions of bagasse (as received), acid pretreatedfiber and hemicellulose hydrolysate were determined as previouslydescribed (Geddes et al., 2010, incorporated herein by reference).Moisture content was measured using a Kern model MLB 50-3 moistureanalyzer (Balingen, Germany) per manufacture's instructions. Sugars,organic acids and furans were measured by high-performance liquidchromatography (HPLC) using an Agilent Technologies 1200 series HPLCsystem (Santa Clara, Calif.) using routine methods.

Statistical Analysis

Graphpad Prism (Graphpad Software, San Diego, Calif.) was used to deriveequations that simulate various relationships. This program was alsoused to perform two-way ANOVA (analysis of variance) of compositionalanalysis using the two-tailed student t-test. Differences in means werejudged significant when P values for the null hypothesis were 0.05 orless.

Example 2 Composition

Bagasse samples were analyzed for carbohydrate composition over a 2-yearperiod before and after acid pretreatment (1% phosphoric acid, 1 h, 145°C.). Sugar compositions are expressed as g/kg dry weight (Table 1).Steam pretreatment with dilute phosphoric acid-solubilized an average of360 g/kg bagasse dry weight. Analysis of the acid pretreated fiberconfirmed that the hemicellulose had been selectively solubilized.Differences in composition were judged significant for all sugars exceptgalactose (p<0.05). As expected, glucan content of the insoluble fiberwas increased by acid pre-treatment. Due to the mild conditions used,approximately 19% of the xylose, 38% of the galactose and 15% of thearabinose remained associated with acid pretreated fiber.

Hemicellulose syrups were separated from acid pretreated fiber and alsoanalyzed (Table 1). These contained 35 g/L total sugar and lowconcentrations of potential inhibitors of fermentation (4.6 g/L acetate,0.5 g/L furfural and 0.3 g/L formate). Soluble sugars recovered in thehydrolysate represented 21% of the initial bagasse dry weight.

TABLE 1 Sugar composition of sugarcane bagasse, washed acid pretreatedfiber and hemicellulose hydrolysate. Material Glucose Xylose GalactoseArabinose^(a) Total sugars Bagasse as 387 ± 20 212 ± 20 26 ± 11 34 ± 26659 ± 40 received^(b) (g/kg) Washed fiber 593 ± 17  64 ± 19 15 ± 8   8 ±10 680 ± 36 after pretreatment^(c) (g/kg) Hemicellulose  4 ± 1 27 ± 2 1± 1 3 ± 1 35 ± 2 hydrolysate^(d) (g/L) ^(a)Arabinose may also includelow levels of mannose and fructose which co-elute. ^(b)mean ± SD (n =14) ^(c)mean ± SD (n = 18) ^(d)Inhibitors present in hemicellulosehydrolysate included (g/L): furfural (0.49 ± 0.10),hydroxymethylfurfural (0.02 ± 0.04), formate (0.27 ± 0.04), and acetate(4.58 ± 0.23). An average of 36% of the bagasse dry weight wassolubilized by acid pretreatment. mean ± SD (n = 54)

Example 3 Effect of Cellulase on Relative Viscosity

The effect of incubation time on viscosity was examined using threecellulase enzyme loadings (0.25 FPU, 0.5 FPU and 5.0 FPU/g dry weightacid pretreated fiber). For the highest enzyme loading, the reduction inrelative viscosity was nearly complete after only 1 h (FIG. 1a ). Longertimes were required for lower enzyme loadings. Relative viscositiesdeclined to plateau values that were inversely proportional to the levelof added cellulase, with no tendency to converge during longerincubation times. This plateau of relative viscosity with continuedincubation is similar to saccharification but not well-understood(Warwick et al., 1985). The relationships between viscosity andincubation time for saccharification can be represented by a one phaseexponential decay for each enzyme loading (FIG. 1a ):0.25FPU/g dry weight acid pretreated fiber, y=23674e^(−0.978t)+2196  (1)0.5FPU/g dry weight acid pretreated fiber, y=24366e ^(−1.592t)+1504  (2)5.0FPU/g dry weight acid pretreated fiber, y=25614e^(−4.050t)+255.1  (3)

For Eqs. (1)-(2), y represents the relative viscosity (cP) and t isincubation time (hours). R-squared values were calculated as 0.991,0.989 and 0.975 for 0.25, 0.5 and 5.0 FPU/g dry weight acid pretreatedfiber, respectively, indicating excellent agreement with experimentalresults. Confidence limits have been included for each curve (p<0.05).

Acid pretreated fiber was slurried in hemicellulose hydrolysate tosimulate process conditions in which solids and liquids were notseparated. Acetate and phosphate present in the hydrolysate served asbuffers for pH adjustment. Although the tangled mass of fiber is farfrom the ideal solutions described by viscosity theory, measurements ofrelative viscosity can provide useful information regarding changes influid properties. Preliminary experiments were conducted with a varietyof cones, paddles and spindles. A small T-bar spindle was found to bethe most useful. Slurries of hydrolysate containing 10% (w/w) acidpretreated fiber were digested with various levels of Biocellulase W(FIG. 1b ). Temperature and pH optima for fungal cellulases(approximately pH 5.0 and 50° C.; Ou et al., 2009) were similar forBiocellulase W (pH 5.0 and 60° C.; Table 2). This demonstrates thatcellulase from any of a number of sources can be used in the methods ofthe invention. Prior to enzyme addition, the T-bar was unable to rotateand registered values exceeding 20,000 cP. The extrapolated initialviscosity (t=0) value from FIG. 1a (i.e., 25,870 cP) was used as amaximum value in this graph. After 6 h incubation with Biocellulase W,relative viscosity was reduced by 77% with an enzyme loading of 0.1FPU/g dry weight acid pretreated fiber, and by 95% with an enzymeloading of 0.5 FPU/g dry weight acid pretreated fiber. Previous studieshave reported that rheological properties of cellulose derivatives arerelated to molecular structure parameters such as molar mass andparticle size (Clasen et al., 2001; Gautier et al., 1991). Viscosityreduction can be accomplished by reducing the molar mass throughenzymatic degradation of polysaccharide chains. Enzymatic treatment ofbiomass disrupts the interaction of fiber polymers such as cellulosechains creating smaller particles, which also decrease the viscosity.The relationship between enzyme loading and relative viscosity (6 hincubation) can be represented by a one phase exponential decay (FIG. 1b):y=247986e ^(−15.75x)+1049  (4)

Here, y represents the relative viscosity in centipoise and x is enzymeloading (FPU/g dry weight acid pretreated fiber). The R-squared valuewas 0.9969, indicating a good fit with experimental results. Confidencelimits have been included for each curve (p<0.05). With this equationthe viscosity of the acid pretreated fiber slurry (10% solids) can beestimated for any enzyme loading (6 h incubation).

TABLE 2 Effects of cellulase enzymes on enzyme-solubilized sugars andrheological properties Enzyme- How Testing Solubilized Treat- (funnelstem Sugars (% Relative Cellulase levels ment diameter dry weight Vis-(FPU/g dry weight) and Time in mm)* acid pre- cosity incubationconditions (h) 2 7 treated fiber) (cP) Effect of Enzyme .0 2 0.0 ±0.0 >20000 Loading (FPU/g .05 2 1.8 ± 0.3 >20000 dry weight acid .1 22.0 ± 0.0 >20000 pretreated fiber, .25 2 4.1 ± 0.8 6000 pH 5, 55° C.) .52 4.9 ± 0.3 1500 .0 2 12.9 ± 1.2  400 .0 6 0.0 ± 0.0 >20000 .05 6 2.5 ±0.0 >20000 .1 6 3.5 ± 0.2 6000 .25 6 5.4 ± 0.4 2300 .5 6 6.8 ± 0.1 1300.0 6 17.6 ± 0.4  200 Effect of pH (55° .0 2 1.4 ± 0.1 >20000 C., 0.5FPU/g .0 2 3.7 ± 0.2 7500 dry weight acid .0 2 8.5 ± 3.4 1500 pretreatedfiber) .5 2 ND 2000 .0 2 6.5 ± 1.6 6000 .5 2 ND >20000 .0 2 1.9 ±0.5 >20000 .0 2 0.5 ± 0.6 >20000 .0 6 3.2 ± 0.1 5000 .0 6 5.9 ± 0.1 1400.0 6 8.4 ± 2.1 500 .5 6 D D D ND ND .0 6 6.7 ± 0.1 600 .5 6 D D D ND ND.0 6 0.7 ± 0.4 >20000 .0 6 0.5 ± 0.4 >20000 Effect of Temper- 5 2 0.9 ±0.1 >20000 ature (° C., 0 2 2.3 ± 0.1 15000 pH 5, 0.5 FPU/g 0 2 3.6 ±0.0 5000 dry weight acid 5 2 4.9 ± 0.3 1500 pretreated fiber) 0 2 5.8 ±0.3 2000 0 2 4.5 ± 0.1 >20000 0 2 1.0 ± 1.0 >20000 5 6 1.4 ± 0.3 >200000 6 4.1 ± 0.3 8000 0 6 7.5 ± 0.1 3000 5 6 11.8 ± 0.4  1700 0 6 D D D NDND 0 6 D D D ND ND 0 6 2.0 ± 0.1 >20000 *The N, Y and ND indicate noflow through the funnel, flow through the funnel, and data that was notdetermined respectively.

Example 4 Effect of Cellulase Loading on Extent of Saccharification

Surprisingly little saccharification was required to reduce viscosity(FIGS. 1b, 1c, and 1d ). After 6 h, a very low enzyme loading of 0.1FPU/g dry weight acid pretreated fiber (3.5% of the fiber dry weightsolubilized) reduced relative viscosity by 77% (FIG. 1b ). With 5.0FPU/g dry weight acid pretreated fiber (6 h), viscosity was reduced by99% accompanied by the saccharification of 17.6% of the dry weight. Therelationship between enzyme loading and saccharification (FIG. 1b ) andthe time course for saccharification (FIG. 1c ) can be represented by afourth and third order polynomials, respectively. Confidence limits havebeen included for each curve (p<0.05).

Extent of saccharification versus enzyme loading (6 h incubation; FIG.1b ):y=0.09071+49.80x−161.9x ²+194.7x ³−32.84x ⁴  (5)

In this equation, y represents the amount of solubilized sugars (% dryweight acid pretreated fiber) and x is enzyme loading as FPU/g dryweight acid pretreated fiber. The R-squared value is 0.9980, indicatingan excellent agreement with experimental results. The enzyme loadingrequired for a desired sugar concentration (6 h of incubation) can beestimated using Eq. (5).

Extent of saccharification versus time using 5.0 FPU/g dry weight acidpretreated fiber (24 h; FIG. 1c ):y=0.6496+7.520t−0.8492t ²+0.02458t ³  (6)

Extent of saccharification versus time using 0.5 FPU/g dry weight acidpretreated fiber (24 h; FIG. 1c ):y=0.1498+2.414t−0.1735t ²+0.003901t ³  (7)

Extent of saccharification versus time using 0.25 FPU/g dry weight acidpretreated fiber (24 h; FIG. 1c ):y=0.01837+1.939t−0.1452t ²+0.003399t ³  (8)

For Eqs. (6)-(8), y represents the amount of enzyme-solubilized sugars(% dry weight acid pretreated fiber) and t is time of enzymaticsaccharification (hours). The R-squared values are 0.99 for all threeenzyme loadings (5.0, 0.5, and 0.25 FPU/g dry weight acid pretreatedfiber), indicating excellent agreement with experimental results. Usingthese equations, the amount of sugar that will be solubilized by aspecified enzyme loading and incubation time (≦24 h) can be estimated.Similar trends were observed for individual sugars (FIG. 1d ). Under themild treatment conditions used, part of the hemicellulose remainedassociated with the fiber (Table 1). This hemicellulose was solubilizedduring incubation with Biocellulase W consistent with the presence ofadditional enzymatic activities (FIG. 1d ). Curves defining individualsugars were not modeled.

Example 5 Effect of Enzyme Treatment on Flow Through Graded Funnels

The handling and transferring of fibrous slurries represent significantchallenges for LCB conversion to fuels and chemicals. We have used threedifferent funnels with internal stem diameters of 7 mm, 12 mm and 17 mmto compare the flow properties of acid pretreated fiber slurries (10%w/w). Flow was tested before and after enzyme treatments (Table 2). Acidpretreated fiber slurries failed to flow through all funnels prior toenzyme treatment. After 2 h of incubation, all enzyme concentrationsallowed the fiber slurries to flow through the 17 mm stem even thoughviscosity measurements were above the measurable range (≧20,000 cP) insome cases. Only the two highest enzyme concentrations (0.5 and 5.0FPU/g dry weight acid pretreated fiber) permitted flow through the 12 mmstem. None permitted flow through the 7 mm stem (2 h). After 6 h ofincubation, the highest enzyme concentration permitted flow through the7 mm stem. Flow properties followed the same trends (pH, temperature,enzyme dosage, time) observed for relative viscosity in most cases, andwere improved by greater saccharification and longer incubation times.Flow through the 17 mm, 12 mm and 7 mm stems occurred at relativeviscosities of ≧20,000 cP, ≦3,000 cP and ≦200 cP, respectively. Thesecorresponded to the saccharification of approximately 1%, 5% and 17% ofacid pretreated fiber.

More practical tests were conducted with acid and enzyme treated bagasseusing a centrifugal pump (Jabsco, White Plains, N.Y.; Model18690-0000,115 V; 7.2 AMPS; 1½ in. inlet diameter, ¾ in outlet) and apneumatic diaphragm pump (IDEX Aodd Inc., Mansfield, Ohio; SandpiperModel SIF Metallic Design Level 1; 1 in. inlet and outlet diameter).Positive results for flow through the 12 mm funnel stem were found to bean excellent predictor of successful pumping. Enzyme dose and treatmentconditions can be used in combination to improve flow properties andpumping of acid pretreated sugarcane bagasse.

Example 6 Correlation Between Extent of Saccharification and RelativeViscosity

Extent of saccharification and relative viscosity were measured under avariety of conditions using six independent samples of bagasse. Thisdata has been assembled into a scatter plot (FIG. 2). Relative viscositywas dramatically reduced by a small amount of saccharification. With 5%saccharification, relative viscosity was reduced by almost 90%. However,saccharification of 20-30% of the dry weight was required to achieve thelowest viscosity. An equation was developed to model this data (FIG. 2).

The decline in viscosity during saccharification was represented by aone phase exponential decay (R-squared=0.9316). Confidence limits werealso included (p<0.05). Eq. (9) can be used to estimate the viscosity ofthe slurry based on enzyme-solubilized sugar, a property that can becorrelated with pumping (Table 2).y=25319e ^(−0.4412x)+592.2  (9)

In this equation, y represents the relative viscosity in centipoise andx is solubilized sugar as a percentage of acid pretreated fiber. Arelative viscosity of 3,000 cP or less was needed for flow through afunnel with a 12 mm ID stem (Table 2) and is indicated by a horizontaldashed line on FIG. 2. Reduction of viscosity to this level required thesolubilization of at least 50 g sugar/kg acid pretreated fiber.

Example 7 Effect of Blending Acid Pretreated Fiber (No Enzyme Digestion)with Enzyme-Digested Acid Pretreated Fiber (pH 5.0, 55° C., 6 h) onViscosity

The physical appearance of acid pretreated fiber slurries wasdramatically altered by enzyme treatments. Initially, slurries with 10%fiber occupied the entire volume, poured as a single tangled unit andfailed to settle indicating extensive bridging and strong interactionsbetween fibers or between fibers and water. After enzyme digestion,slurries readily settled and behaved as a suspension of independentparticles with lower viscosities.

Additional experiments were conducted to determine the effect ofcombining enzyme-digested slurries of acid pretreated fiber andundigested slurries on the relative viscosity of the mixture (FIG. 3).Acid pretreated fiber slurries (10% dry weight fiber in hydrolysate)were digested for 2 h (FIG. 3a ) or 6 h (FIG. 3b ) using differentamounts of Biocellulase W (5.0, 0.5, and 0.25 FPU/g dry weight acidpretreated fiber) and cooled to room temperature to minimize furtherenzyme action. Relative viscosity was measured immediately after mixing(within 1 min) with various amounts of acid pretreated fiber (no enzymetreatment). Relative viscosities of all enzyme treated bagasse were low(200-2,500 cP) in comparison to undigested material (≧20,000 cP).Addition of small amounts of acid pretreated fiber (no enzyme digestion)had little effect on relative viscosity until a threshold concentrationwas reached (FIG. 3). At this point, further addition of acid pretreatedfiber resulted in an abrupt increase in viscosity. The proportion ofacid pretreated fiber (no enzyme digestion) that could be accommodatedbelow each threshold value varied considerably (5-35%) and was directlyrelated to the initial viscosity of enzyme-digested bagasse. Using 3,000cP as a conservative maximum for pumping based on funnel experiments (12mm stem), up to 35% fresh acid pretreated fiber could be blended withenzyme-digested acid pretreated fiber (5.0 FPU/g dry weight acidpretreated fiber; 6 h). At one-tenth this enzyme loading, up to 23%undigested acid pretreated fiber could be accommodated. At 0.25 FPU/gdry weight acid pretreated fiber cellulase loadings, the addition ofmore than 5% to 6% undigested acid pretreated fiber resulted in adramatic rise in viscosity. The relationship between acid pretreatedfiber additions (fresh) to enzyme-digested fiber and relative viscositycan each be represented by equations (10)-(12) sigmoidal curves) foreach level of enzyme (FIG. 3b ).

0.25 FPU/g dry weight acid pretreated fiber (6 h incubation):

$\begin{matrix}{y = {1324 + \frac{874046}{1 + 10^{({3.30 - {0.067\; x}})}}}} & (10)\end{matrix}$

0.5 FPU/g dry weight acid pretreated fiber (6 h incubation):

$\begin{matrix}{y = {224.3 + \frac{313535.7}{1 + 10^{({2.70 - {0.028\; x}})}}}} & (11)\end{matrix}$

5.0 FPU/g dry weight acid pretreated fiber (6 h incubation):

$\begin{matrix}{y = {811.6 + \frac{645898.4}{1 + 10^{({5.67 - {0.091\; x}})}}}} & (12)\end{matrix}$

In Eqs. (10)-(12), y represents the relative viscosity (cP) and x is thefraction of undigested fiber as a percentage of total (10% dry weight).The R— squared values are 0.9795, 0.9461 and 0.9946 for the equationsabove respectively, indicating excellent agreement with experimentalresults. The large confidence limits for sigmoidal curves obscured thecurves and were omitted. Two-hour treatments were judged to have too fewdata plots to develop a model (FIG. 3a ).

These data indicate that acid pretreated fiber that has been partiallydigested with enzymes has the ability to accommodate or passivateadditions of fresh acid pretreated fiber (no cellulase treatment; up to30% of total fiber) with little increase in viscosity. Without beingbound by mechanism, this observation, together with the rapid decline inviscosity resulting from limited saccharification suggests a simplemechanism. It is suggested that the viscosity of acid pretreated fiberis proposed to result from the tangling interactions of surface-exposedmicro-fibers as observed previously and the bonding between fibrils andwater (water-holding capacity) in the amorphous regions of fibers.Digesting these small fibers with enzymes provides a smooth surface,reducing viscosity with limited saccharification. These smoothenzyme-treated particles would also serve as a diluent that physicallyhinders associations between small fibers on the surface of fresh acidpretreated fiber (no enzymes) until the threshold concentration forrandom associations is reached leading to increased viscosity (FIG. 3).

Example 8 Modeling an Ideal Continuous Stirred Tank Reactor (CSTR) toDecrease Viscosity

We have used the experimental data (FIGS. 2 and 3) to estimate the upperbound viscosity of an ideal CSTR for liquefaction at different meanresidence times (r). The residence time distribution (RTD) of thereactor is a probability function describing the length of time thefluid elements of the tank spend inside the reactor. The ideal CSTRassumes that the material at the inlet is instantly and completely mixedinto the bulk material of the reactor and that the contents of thereactor have the same composition as the outlet at all times. The idealCSTR has an exponential residence time distribution (E(t); Fogler,1986):

$\begin{matrix}{{E(t)} = {\frac{1}{\tau}{\mathbb{e}}^{{- t}/\tau}}} & (13)\end{matrix}$

Here τ represents the mean residence time, defined by τ=V/Q where V isthe volume of the tank and Q is the inlet volumetric flow rate, and τrepresents residence time. The fraction of the reactor contents that hasa retention time between t and t+dt inside the reactor is given byE(t)dt. The fraction of the reactor contents that has a retention timeless than t₁ and greater than t₁ are given by Eqs. (14) and (15)respectively (Fogler, 1986).∫₀ ^(t) ¹ E(t)dt  (14)∫_(t) ₁ ^(∞) E(t)dt=1−∫₀ ^(t) ¹ E(t)dt  (15)

Using Eqs. (13)-(15) above, the fraction wi of the reactor contents witha residence time t such that iΔt≦t<(i+1)Δt can be expressed as:

$\begin{matrix}{w_{i} = {\left( {1 - {\mathbb{e}}^{\frac{{- \Delta}\; t}{\tau}}} \right)*{\mathbb{e}}^{\frac{{- i}\;\Delta\; t}{\tau}}}} & (16)\end{matrix}$

If we make the assumption that viscosity is additive, then the viscosity(μ) of the reactor contents can be expressed in terms of the viscosityμ_(i) of fraction i:

$\begin{matrix}{\mu = {\sum\limits_{i = 0}^{\infty}\;{w_{i}\mu_{i}}}} & (17)\end{matrix}$

However, when FIG. 3's data is plotted with Eq. (17) for each enzymeloading, the experimental data fall below the linear viscosity curve(Eq. 17) until a maximum viscosity value, μmax, is reached:

$\begin{matrix}{\mu \leq {\sum\limits_{i = 0}^{\infty}\;{w_{i}\mu_{i}\mspace{14mu}{if}\mspace{14mu}\mu}} < \mu_{\max}} & (18)\end{matrix}$

From Eqs. (1)-(3) we obtain:μ_(i) =a+be ^(−icΔt)  (19)

where a, b, and c are constants derived from the best fit model of theexperimental data (FIG. 1a ) for each enzyme loading using theconstraint that the initial viscosity for undigested material at t=0 isthe same for all three enzyme loadings. This constraint allowed thederivation of a relative viscosity value (i.e. 25,870 cP) for undigestedmaterial, which was not measurable with the instrumentation but could beused to model an ideal CSTR.

By substituting Eq. (19) for μi and Eq. (16) for wi into Eq. (18) weobtain:

$\begin{matrix}{\mu \leq {\sum\limits_{i = 0}^{i = \infty}\;\left( {\left( {a + {b\;{\mathbb{e}}^{{- {ic}}\;\Delta\; t}}} \right)*\left( {1 - {\mathbb{e}}^{\frac{{- \Delta}\; t}{\tau}}} \right)*{\mathbb{e}}^{\frac{{- i}\;\Delta\; t}{\tau}}} \right)}} & (20)\end{matrix}$

which can be expressed as:

$\begin{matrix}{\mu \leq {a + {b\frac{1 - {\mathbb{e}}^{\frac{{- \Delta}\; t}{\tau}}}{1 - {\mathbb{e}}^{{- {({c + \frac{1}{\tau}})}}\Delta\; t}}}}} & (21)\end{matrix}$

Taking the limit Δt→0 we get 0/0, so we can use L'Hopital's Rule:

$\begin{matrix}{\mu \leq {a + {b_{{\Delta\; t}\rightarrow 0}^{\lim}\frac{{- {\mathbb{e}}^{\frac{{- \Delta}\; t}{\tau}}}*\left( {- \frac{1}{\tau}} \right)}{{- {\mathbb{e}}^{{- {({c + \frac{1}{\tau}})}}\Delta\; t}}*\left( {- \left( {c + \frac{1}{\tau}} \right)} \right)}}}} & (22) \\\left. \Rightarrow{\mu \leq {a + \frac{b}{1 + {c\;\tau}}}} \right. & (23)\end{matrix}$

Eq. (23) can be used to estimate an upper bound viscosity in a CSTR witha specific residence time and enzyme loading when the viscosity fallsbelow a certain μmax. The maximum viscosities we could establish anupper bound for (i.e. μmax) at each enzyme loading were determinedgraphically by transposing Eq. (17) with Eqs. (10)-(12) and were 5,500cP, 12,000 cP, and 11,200 cP for 0.25 FPU, 0.5 FPU, and 5 FPUrespectively. Predicted upper bound values for viscosity are given inTable 3. From this model, the predicted mean residence times to producea slurry that can be pumped (i.e., viscosity≦3,000 cP) are 30 h, 9 h,and 2 h for 0.25 FPU, 0.5 FPU, and 5 FPU respectively.

TABLE 3 Predicted upper bound viscosity for an ideal continuous stirredtank reactor (CSTR). Enzyme loading Viscosity at different meanresidence times (τ, hours) (FPU/g fiber) 0.5 1 2 3 4 5 6 7 8 9 10 20 300.25 18096 14166 10206  8214 7016 6216 5643 5213 4879 4611 4392 33472976 0.50 15069 10903 7326 5721 4810 4222 3812 3510 3277 3093 2943 22452003 5.00  8723  5327 3069 2203 1744 1461 1268 1128 1022 939 872 568 464Note: Italicized values are above the confidence limit (i.e. viscosity >μ_(max)) and therefore cannot be used as upper bound viscositypredictions.

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Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of decreasing the relative viscosity offeedstock comprising: incubating the feedstock with about 0.01 FPU toabout 20 FPU cellulase/g dry weight of feedstock for about 10 minutes toabout 10 hours at a pH of about 2 to about 6 at a temperature of about40° C. to about 80° C., said feedstock, prior to treatment, having arelative viscosity of at least 20,000 cP; thereby decreasing therelative viscosity of the feedstock.
 2. The method of claim 1, whereinthe feedstock is a bagasse, corn fiber, corn stover, a plant wastematerial, a processing or agricultural byproduct, a sugar cane,monoenergy cane, sorghum sudan, Miscanthus, switchgrass, wheat, milo,bulgher, barley, rice, corn, beet, or tree.
 3. The method of claim 1,wherein the feedstock is incubated with about 0.05 FPU to about 20 FPUcellulase/g dry weight of feedstock.
 4. The method of claim 1, whereinthe feedstock is incubated for about 10 minutes to about 6 hours.
 5. Themethod of claim 1, wherein the relative viscosity after treatment isless than 8000 cP.
 6. The method of claim 1, wherein the pH is about 3to
 6. 7. The method of claim 6, wherein the feedstock is incubated forabout 10 minutes to about 6 hours.
 8. The method of claim 6, wherein therelative viscosity after treatment is less than 8000 cP.
 9. The methodof claim 1, wherein the feedstock is incubated at a temperature of:about 45° C. to about 80° C.
 10. The method of claim 9, wherein thefeedstock is incubated for about 10 minutes to about 6 hours.
 11. Themethod of claim 9, wherein the relative viscosity after treatment is:less than 8000 cP.
 12. The method of claim 1, wherein the feedstock ispretreated in an acidic solution prior to incubation with cellulase. 13.The method of claim 12, wherein the acidic solution is phosphoric acidor sulfuric acid.
 14. The method of claim 13, wherein the phosphoricacid solution comprises about 0.01% (w/w) to about 10% (w/w) phosphoricacid.
 15. The method of claim 13, wherein the sulfuric acid solutioncomprises about 0.01% (w/w) to about 10% (w/w) sulfuric acid.
 16. Themethod of claim 12, wherein the feedstock is pretreated in an acidicsolution for about 2 minutes to about 2 hours.
 17. The method of claim1, wherein the feedstock is pretreated with steam for about 1 hour toabout 3 hours prior to treatment with cellulase.
 18. The method of claim17, wherein the feedstock is treated with steam after treatment with anacidic solution.
 19. The method of claim 18, wherein the feedstock priorto treatment comprises a mixture solution of about 8-12% dry weight inan aqueous solution.
 20. The method of claim 1, wherein the methoddecreases relative viscosity of the feedstock by at least 50% ascompared to the starting material and/or extracts at least about 40% offermentable sugars found in the feedstock.
 21. The method of claim 20,wherein the fermentable sugars comprise one or more of xylose,galactose, and arabinose.
 22. The method of claim 1, wherein said methodextracts fermentation inhibitors from said feedstock in an amount lessthan 1% of the volume of the incubation mixture formed by the mixture offeedstock with cellulase.
 23. The method of claim 22, wherein thefermentation inhibitors comprise one or more of furfural,hydroxymethylfurfural, formate, and acetate.
 24. A method of decreasingthe relative viscosity of feedstock comprising: incubating the feedstockwith about 0.50 FPU to about 5 FPU cellulase/g dry weight of feedstockcellulase for about 15 minutes to about 2 hours; wherein the feedstockis incubated at a pH of about 3 or less at a temperature of at least 60°C. and the feedstock, prior to treatment, has a relative viscosity of atleast 20,000 cP; thereby decreasing the relative viscosity of thefeedstock and the relative viscosity after treatment is less than 1500cP.
 25. A method of reducing the viscosity of a feedstock having aviscosity of at least 20,000 cP comprising: combining the feedstockhaving a viscosity of at least 20,000 cP with a feedstock obtained bytreating a feedstock by the method of claim 1, wherein the feedstockhaving a viscosity of at least 20,000 cP comprises 30% or less of thevolume of the feedstock treated by the method of claim 1.